This proposal addresses a key issue related to the pathogenesis of HIV-1 infection and the design of immunotherapeutic strategies based on cytotoxic T lymphocytes (CTL). CTL play an important protective role in HIV-1 infection, but viral sequence plasticity and mutations in the sequences they recognize (epitopes) limit their efficacy. However, two factors oppose viral escape mutation: 1) limitations of the ability of HIV-1 to tolerate required mutations for escape (fitness constraints), and 2) the ability of CTL to recognize many epitope sequence variants. This project will explore and exploit these mechanisms by defining the epitope sequence options for escape from CTL, and establishing strategies to expand the CTL coverage of these epitope variants. This work offers both better understanding of CTL escape (comparing CTL responses that are more versus less prone to escape in vivo) as well as development of strategies that could be applied to novel gene therapeutic approaches to create superior CTL responses. The overall approach will be to examine the T cell receptors (TCR) of CTL that recognize HIV-1 epitopes, and the epitope variants that are recognized or not recognized. Thus, each TCR will have its subset of recognized epitope variants defined, and the collection of TCR in a person will define the total coverage of epitope variation in vivo. Across several persons recognizing an epitope, there will be the potential to combine nonoverlapping TCR in a manner to minimize possibilities for epitope escape mutation. Panels of TCR to avoid epitope escape mutation would be useful for currently developing TCR gene therapy approaches for HIV treatment. The specific aims are: 1) To isolate TCR for CTL responses against immunodominant epitopes presented by protective HLA types (which have been shown to have limited options for epitope mutation and escape), and well-defined immunodominant epitopes presented by common HLA types;2) To define the patterns of epitope variant recognition for these TCR against HIV-1-infected cells;3) To assemble panels of TCR that minimize available pathways for HIV-1 escape. This project therefore addresses the recent focus of NIAID on basic pathogenesis research with translational applications to novel therapeutics or prevention for HIV infection.